The present invention relates to a method and a device for lysing bacteria or eukaryotic cells, as well as for extracting and purifying nucleic acids, and in particular plasmids, from bacteria or eukaryotic cells containing these plasmids.
The production of plasmids of interest, and in particular of plasmids into which a gene or a coding sequence of DNA has been inserted, is achieved by making a multicopy of these plasmids in bacteria capable of producing a large number of these plasmids, and in particular in certain strains of Escherichia coli which are high plasmid producers and which are often already used in the laboratory or on an industrial scale.
One of the applications which requires major production of a plasmid, on an industrial scale, is the manufacture of medicaments or vectors of prophylactic or therapeutic interest based on a naked plasmid or a plasmid combined with means for penetrating into the cells of the recipient host. Such applications are described for example in Patent Application WO 90/11092.
Several techniques exist for bacterial lysis which allow the extraction of the plasmids, followed by a separation, and therefore by a purification of these plasmids. The technique most commonly used is the alkaline lysis technique which uses an alkaline lysing agent such as a sodium hydroxide+SDS (sodium dodecyl sulphate) preparation, followed by neutralization with an acidic agent such as potassium acetate. This neutralizing agent also has the effect of precipitating all the bacterial constituents including the genomic DNA, the supernatant containing essentially the plasmid DNA. The supernatant may then be separated from the precipitate by centrifugation or filtration (Birnboim Methods In Enzymology (1983) 100:243). The alkaline lysis technique is most particularly recommended for lysing bacteria; however, it can be used equally well for lysing eukaryotic cells.
This technique, which is suitable on a laboratory scale, is difficult to carry out on an industrial scale because the lysate resulting from the action of the alkaline agent on the bacteria constitutes a very viscous suspension, such that the lysate being formed has to be homogenized by stirring. Likewise, after neutralizing with acid, homogenization by stirring is required. This stirring is delicate and it is generally carried out manually, the operator gently stirring the bottles or flasks containing the lysis medium. An insufficient stirring results in a lysis of poor quality whereas an excessive stirring tends to fragment the genomic DNA, which subsequently mixes with the plasmids. In both cases, a reduction in the plasmid DNA yield is observed. Consequently, the dexterity of the operator is an essential requirement for the success of the operation. In order to automate the method of lysis, it has already been proposed in the document WO 97/23601 to continuously pass the suspension of cells to be lysed and a solution of a lysing agent through a static mixer of sufficient length in order to complete the lysis. The lysate resulting therefrom may then be conveyed through a tubing to a second static mixer, into which a solution of the precipitating agent also enters.
Such a method therefore uses a specific means, namely a static mixer, which replaces the manual stirring of large volumes by a continuous stirring over the whole length of the mixer. The use of such a mixer, in addition to requiring the purchase, maintenance and cleaning of the device, requires a fixed duration of contacting with the lysing agent, with no practical possibility of controlling it or of varying it. Furthermore, the stirring maintained in the mixer, even if it is preferable to the poorly controlled mixing of batch volumes, can cause breaks in or degradation of cellular constituents and in particular of nucleic acids.
Another method, described for example in the document WO 96/36106, also makes it possible to establish a continuous lysis, but this time without using stirring means. This method consists (i) in preparing a mixture of a bacterial suspension and of an agent, such as lysozyme, in incubating this mixture for about one hour in order to make the bacterial wall fragile, and then (ii) in passing a stream of this mixture inside a tubing heated to a high temperature (70-100xc2x0 C.). The action of the heat promotes the lysis. The disadvantage of such a solution is to require a means of heating at high temperatures and adjusting the temperature to the various specific cases which may be encountered.
The present invention is intended to overcome these disadvantages and to provide a method of cell lysis which is applicable in particular to the extraction and purification of nucleic acids such as plasmids from bacteria or eukaryotic cells, and which is capable of being used without manual intervention and under extremely inexpensive conditions.
Another objective of the invention is to provide a method which makes it possible to control extremely precisely the conditions and the duration of the lysis and of the extraction, this being for all the cells present.
Another objective is to provide a method which makes it possible to establish substantially homogeneous lysis conditions for a population of cells.
Another objective is to provide a method capable of being used in a closed medium, protected from contaminations, which is an advantage for the pharmaceutical quality of the desired products, for example plasmids.
Another objective is to obtain a reduction in the duration of the cell/lysing agent contact which is necessary for completing the lysis.
Another objective is to provide a device for carrying out this method, a device which is simple and not very expensive.
Another objective is to provide on the industrial scale plasmid preparations with a high and substantially enhanced yield compared to that for preparations which may be obtained according to the prior art methods.